A technology to induce differentiation of pluripotent stem cells holds the key for realization of regenerative medicine and establishment of in vitro drug screening study or evaluation of drug safety. In particular, it is important for the generative medicine and drug evaluation for heart diseases because heart diseases are currently the second cause of death in Japan. There are various drugs which induce severe side effects, including cardiac arrest and arrhythmia, leading to increasingly-demand to provide homogenous cardiac muscle cells which are useful for cardiotoxicity study. So far, it has been reported that cardiac muscle cell differentiation of human embryonic stem (ES) cells is induced by co-culturing human ES cells and mouse feeder cells, END2 cells (Mummery, C., et al., “Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells.” Circulation. 107(21), 2733-40 (2003); incorporated herein by reference in its entirety). However, differentiation efficiency of this method is not satisfactory and it is difficult to obtain pure human cardiac muscle cells since the resulting human cardiac muscle cells are often contaminated with mouse END2 cells. It is also reported that cardiac muscle cell differentiation is induced by preparing embryoid from ES cells and adding several cytokines (fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4), vascular endothelial cell growth factor (VEGF), Dickkopf-1 (DKK1), Activin A) to the embryoid (Yang, L., et al., “Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population,” Nature, 453(7194), 524-8 (2008); Leschik, J., et al., “Cardiac commitment of primate embryonic stem cells.” Nat. Protoc. 3(9), 1381-7 (2008); each incorporated herein by reference in its entirety). This method, however, requires a large amount of cytokines and can be rather expensive, while its differentiation efficiency is not enough.
There is therefore a continuing need in the fields of regenerative medicine and drug discovery to develop methods that can induce differentiation of pluripotent stem cells into cardiac muscle cells with high efficiency, minimal contamination and yet be cost-effective and suitable for commercial production.